Various approaches are known from the prior art for increasing the lateral and axial resolution of such laser scanning microscopes even beyond the diffraction limit of the illumination light, and correcting imaging errors in the process.
Initially, a distinct contrast and resolution improvement was attained with confocal scanning microscopy. In so doing, a laser is used, which illuminates an object in the focal plane and excites fluorescence molecules at all points there. The fluorescence light is imaged on a pinhole in the image plane, only the light directly reaching the focal plane being detected. With confocal imaging, the lateral resolution (which is still diffraction-limited) can be greater by a factor of 1.4 than in conventional microscopy. This depends on the size of the pinhole. For this reason, the size of the pinhole must be optimized here to the size of the spot to be imaged. A pinhole that is too small reduces the amount of usable light, too large a pinhole leaves too much light outside the focal plane and admits too much scattered light.
Methods like InM and 4Pi microscopy improve the axial resolution through the use of two objective lenses with high numerical aperture and superposition of the images in the detection plane either by wide field or confocal laser fluorescence configuration, or by the use of multiple excitation light sources, patterns due to interference being projected onto the sample. The lateral resolution remains unchanged in the process.
Other scanning high-resolution methods are RESOLFT (reversible saturable optical (fluorescence) transitions) microscopy, wherein especially sharp images are obtained. Instead of using conventional objective lenses and diffracted beams, a resolution far beyond the diffraction limit is obtained, down to the molecular scale. RESOLFT microscopy overcomes this diffraction limit by temporarily switching the dyes into a condition wherein they are no longer able to respond with a (fluorescence) signal following illumination.
Special methods of non-scanning optical microscopy, more precisely of fluorescence microscopy, are known as the PALM (photoactivated localization microscopy) method or the STORM (stochastic optical reconstruction microscopy) method. They rely on light-controlled on-and-off switching of fluorescence in individual molecules. In the process, switching on and off is accomplished beyond a certain time interval, during which several individual images can be taken. By means of a subsequent computer calculation, the position of individual molecules can be defined with a resolution beyond the optical resolution limited described by Ernst Abbe.
A microscopy method with increased resolution is known from EP 2 317 362 A1, wherein the illumination or the illumination pattern is shifted, with respect to the detection, with an accuracy exceeding the achievable optical resolution, and several images are taken and evaluated during the shift.
Moreover, it has long been known to use adaptive optics, that is, optically active components, for wavefront modulation. The adaptive optics deliberately alters the phase and/or the amplitude of the light in such a manner that both shifting and forming of the focus in the space, as well as correction of aberrations, if any, can be accomplished. An axial shift of the is achieved by a change of the wavefront. Here, an axial shift of the focus corresponds to a spherical change of the wavefront, a lateral shift to tilting of the wavefront. Aberrations in the beam path are also compensated by changing the wavefront. These manipulations are carried out in an aperture plane of the beam path with the aid of deformable mirrors.
Such adaptive optics systems are described for example in EP 1 253 457 B1, US 7 224 23 B2 or JP 2008 026643 A.
The use of adaptive optics for correcting, for example, sample-induced wavefront errors (including wavefront errors due to the sample carrier and the immersion medium), is known from a multitude of publications.
The problem in implementing adaptive optics is always that a control signal is required for the adaptive optics. It is also necessary to initially specify the wavefront error before it can be removed. Generally, the wavefront error is not known.
Two solution approaches exist in the prior art: in the first solution, an additional measuring system is brought into the microscope so as to directly measure the wavefront error, e.g. by means of an ordinary and known Shack-Hartmann sensor. US 2004/0223214 A1 shows for example a microscope with a Hartmann-Shack wavefront sensor. From the shape of the wavefront, the aberrations which were caused by scattering of the light in the sample can be defined. Depending on performance (correction degrees of freedom of the correction element), various effects can thus be corrected. In elements with very many degrees of freedom, such as an SLM (spatial light modulator) for example, this corresponds to the number of controllable pixels. With such elements, not only aberrations (slowly varying wavefronts) of the system and of the sample, but also high-frequency components (scattered light) can be corrected, to the extent that these can still be measured at the wavefront sensor. In a particular image segment, depending on the scan position, this sometimes very high-frequency wavefront is thus corrected at the element. Consequently, the diffraction-limited performance of the system can also be attained in media or scattering samples, and particularly also for non-vanishing system aberrations of the microscope.
As a consequence, valuable photons only available in a finite number must be for a sensor; these photons are subsequently no longer available for the actual measurement.
In the second solution, the wavefront error is defined iteratively directly from the LSM signal, i.e. the wavefront is optimized until the LSM signal is optimal. For this purpose, a large number of iteration cycles is needed; 10 to 30 cycles are reported in the literature.
Fluorophors fade and can only emit about 50,000 photons. In both prior art solutions, therefore, the disadvantage arises that many of the few photons must be “sacrificed” for determining the control signal for the adaptive mirror.
In conventional scanning microscopes (other than STED), as a matter of principle, diffraction-limited imaging of the (ideally point-like) spot as a so-called Airy disk takes place, which is defined by a point response or point image function, or point-spread function—PSF. The PSF expresses how an idealized, point-like object is imaged by a system. What is problematic is that suitable sensors capable of imaging the Airy disk in the sub-mm range at the desired resolution are not currently available, and other techniques are either very expensive or very slow and are thus not suitable for commercial use.
In conventional scanning microscopy, the imaging spot is evaluated pixel-wise, i.e. for each spot position exactly one pixel is evaluation or one pixel of the total image results from each spot position. Here, only the overall beam intensity (integral) captured by the detector is defined and converted into grey levels. If applicable, suitable pixel or information superposition can take place for small scanning steps. Contrast improvement can be attained here by increasing the grey-scale resolution (color depth). The PMTs (photomultiplier tube) or PMT arrays employed exhibit internal noise at certain amplification ratios, with the result that signal quality declines.
In all these solutions, the problem typically arises that the aberrations vary at different positions on the sample and with different focus positions. These are unknown for the most part, or must be determined by elaborate measurements.
Due to the limited photon yield in fluorescence microscopy, additional measurement of the actual PSF has not been common (or only in wide-field microscopy) up until now.
In a laser scanning microscope, the theoretical PSF can be interfered with by various deviations of the system from the ideal condition or of the sample to be observed. The signal thus becomes interference-prone (noise, signal-to-noise ratio . . . ) and the maximum resolution is reduced.
An object of the invention is to create a scanning microscope and a method for location-dependent aberration correction in the preferably high-resolution scanning microscope.
This is achieved by a laser scanning microscope with the features of claim 1 and by a method with the features of claim 6.
Advantageous modifications and exemplary embodiments are presented in the sub-claims.
The aberrations depend on various influential quantities. The aberrations of the optics of a scanning microscope are known and can be suitably corrected. Sample- and environmentally-dependent refractive index fluctuations, temperature fluctuations and various cover glass thicknesses vary to that effect according to the sample, but are largely constant for each sample. Properties of typical biological samples are often locally only slowly varying. One exception to this is imaging in deep samples, where stronger location-dependence exists due to scattering and a greater correction effort is consequently required.